System and method useful for sarcomere imaging via objective-based microscopy

ABSTRACT

Biological tissue such as skeletal and cardiac muscle can be imaged by using an objective-based probe in the tissue and scanning at a sufficiently fast rate to mitigate motion artifacts due to physiological motion. According to one example embodiment, such a probe is part of a system that is capable of reverse-direction high-resolution imaging without needing to stain or otherwise introduce a foreign element used to generate or otherwise increase the sensed light. The probe can include a light generator for generating light pulses that are directed towards structures located within the thick tissue. The system can additionally include aspects that lessen adverse image-quality degradation. Further, the system can additionally be constructed as a hand-held device.

This patent document is a divisional under 35 U.S.C. § 120 of U.S.patent application Ser. No. 13/305,390 filed on Nov. 28, 2011 (U.S. Pat.No. 8,897,858), which is a continuation-in-part of U.S. patentapplication Ser. No. 12/165,977 filed on Jul. 1, 2008 (U.S. Pat. No.8,068,899), which claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application Ser. No. 60/947,769 filed on Jul. 3,2007; these patent documents, including the Appendix filed in theunderlying provisional patent application, are fully incorporated byreference.

FIELD OF THE INVENTION

The present invention relates generally to biomedical cellular-levelimaging systems and methods and more specifically to minimally-invasivesystems and methods for characterizing biological thick tissue as afunction of properties that are intrinsic to the tissue.

BACKGROUND

Biomedical-engineering advancements have provided a variety of tools toexplore the detailed structure and behavior of biological tissues.Traditional equipment in this area has provided images and other data byway of x-rays, sound waves, and visible and infrared (IR) light tocharacterize the structure and behavior of certain tissues. Althoughgenerally successful, the image quality provided by such conventionalequipment is limited and not applicable to all types of biologicaltissues. As examples, x-ray equipment typically transmits relativelylow-level radiation and is used to characterize the location of thetissue as a function of its periphery, and visible/IR light imagingtools are used for characterizing transparent and semi-transparenttissue but are ineffective for imaging optically-dense (“thick”) tissue.

Conventional approaches for high-resolution images of thick tissue havenot been widely implemented due to approach-specific issues. Generally,these approaches can be categorized as “transmission-mode” (a.k.a.,“forward-direction”) systems and “reverse-direction” systems.Transmission-mode systems radiate energy at the tissue from one side anduse a nearby sensing device on the opposite side of the tissue to sensethe radiated energy after it is impacted by the tissue. One form offorward-direction imaging relies on SHG (second harmonic generation)which is known to be a forward-directed nonlinear optical process. InSHG, a light source directs photons at a target material for interactingand combining into higher-energy photons. The higher-energy photons arepredominantly forwardly-directed at a sensing device on the oppositeside of the tissue. While useful for many in vitro applications, thistransmission-mode approach can be extremely invasive due to the need fora sensing device on the opposite side of the tissue. In moretissue-sensitive applications such as in vivo examinations and in vitroinvestigations where the integrity of the tissue is to be maintainedafter examination, this approach would be unacceptable due to theplacement of the sensing device deep within the subject underexamination.

Reverse-direction systems radiate energy at the tissue from one side anduse a sensing device on the same side of the tissue to sense energyradiated in response. Unlike transmission-mode systems, these systems donot require placement of a sensing device on the opposite side of thetissue and therefore could be considered less invasive for in vivoapplications. For high-resolution imaging of thick tissue, however,these systems require relatively strong signals and can requirepre-treatment of the tissue with a foreign matter (e.g., dye, exogenousgene or protein) in order to enhance signals responding to excitation ofthe tissue by light. Such pre-treatment is undesirable for reasonsconcerning the invasiveness of the foreign matter and its alteration ofthe cells under examination.

Recent attempts to use reverse-direction systems have not been widelyadopted. These attempts have relied on back-directed SHG or onendogenous (or native) fluorescence for tissue characterization for avariety of reasons. These approaches are burdened by insufficient signalstrengths and/or the need to physically mitigate physiological motionsassociated with blood flow and respiratory activity. For imagingskeletal and/or cardiac muscle tissues, motions associated withsarcomere contractions further perturb image quality.

SUMMARY

The present invention is directed to methods for using and arrangementsinvolving an optical probe for characterizing biological thick tissue.Certain applications of the present invention are directed to overcomingthe above-mentioned limitations and addressing other issues as maybecome apparent in view of the description herein.

The present invention provides significant bio-medical high-resolutionimaging advancements with minimally-invasive optical-probeimplementations that produce high-resolution images of biological thicktissues using predominantly intrinsic biocellular sources. One exampleembodiment of the present invention uses a microendoscopic probeinserted, like a needle, as part of a minimally-invasive imagingprocedure for stimulating structures intrinsic to the thick tissue. Theprobe is also used to collect the resulting signal for characterizationof the tissue structure. The optical probe scans the thick tissue at aline resolution rate that is sufficiently-fast to mitigate motionartifacts due to contractile motion and/or physiological motion. In thiscontext, a high-resolution imaging application produces images atsarcomere-level with subcellular detail and subcellular detail of otherstructures, while mitigating motion artifacts due to contractile motionand/or physiological motion such as respiration and blood flow. Certainexample embodiments are implemented in vivo.

According to a particular embodiment of the present invention, thebio-medical imaging involves a reverse-direction operation. Suchimplementations, in accordance with the present invention, producehigh-resolution images of biological thick tissues using aminimally-invasive optical probe to sense relevant intrinsic signals,thereby avoiding problems associated with pre-treatment of the tissuewith a foreign matter, such as fluorescent dye, exogenous gene orprotein.

According to more specific embodiments, the characterization can be inany of various forms which are sometimes application-dependent and/ordependent on the tissue. For instance, in one specific application, anoptical probe is inserted into skeletal muscle. Light pulses transmittedby the probe stimulate the generation of signals, such as fluorescenceand/or SHG, from intrinsic properties of certain tissue structures.These signals are collected and then processed to provide informationsuch as sarcomere lengths, mobility, and indications of tissuedysfunctionality. This information can be provided in forms includingdisplayed forms, for example, reports, units of measure and biologicalreproduction images, as well as non-displayed forms such as storedelectronic data useful for latent processing.

According to certain example embodiments of the present invention, asystem is implemented for visualizing sarcomeres in vivo. The systemincludes an optical probe having a light-pulse generator to send lightpulses from the optical probe to certain targeted structure in thetissue. A photosensor senses, in response to the light pulses, selectedsignals generated from the sarcomere tissue and predominantly presentdue to properties intrinsic to the targeted structure. A signalprocessor is communicatively coupled to the optical probe tocharacterize the sarcomere tissue based on the sensed selected intrinsicsignals.

In more specific embodiments, the light pulses from the light-pulsegenerator are tuned to a wavelength that interacts with the propertiesintrinsic to the tissue structure. The selected signals are generatedfrom fluorescent mitochondrial molecules or from SHG.

The above summary is not intended to describe each illustratedembodiment or every implementation of the present invention. The figuresand detailed description that follow more particularly exemplify theseembodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be more completely understood in consideration of thedetailed description of various embodiments of the invention thatfollows in connection with the accompanying drawings, in which:

FIG. 1A illustrates an endoscopic imaging system adapted to excitestructure(s) with optical signals (e.g., fluorescent NADH (nicotinamideadenine dinucleotide) or SHG signal) within certain thick tissuestructures and to collect resulting intrinsic signals in response,according to an example embodiment of the present invention;

FIGS. 1B and 1C are images of animal tissue obtained in vivo via amicroendoscopic probe according to an example embodiment of the presentinvention;

FIG. 2 shows a flow diagram depicting a method for use with anendoscopic imaging system adapted to excite optical signals based onintrinsic properties of thick tissue structures and to collect intrinsicsignals in response, according to another example embodiment of thepresent invention;

FIG. 3 shows an example implementation of the present invention forspecific application for high-resolution imaging cardiac sarcomeres,with an optional stimulus step being provided for altering a conditionof the heart and repeating certain steps to provide additional imagingassessing the biological tissue;

FIG. 4A-4D show lead channels that simultaneously deliver electricalleads (commonly used for cardiac stimulus) along with multiple(send/receive) optical fibers, according to various exampleimplementations of the present invention;

FIG. 5 is another endoscopic imaging system in accordance with thepresent invention;

FIGS. 6A-6D are representations of images showing the dynamics ofsarcomere contradictions, also consistent with the present invention;

FIG. 7 is an optical pathway of a microscope, consistent with theinstant disclosure;

FIG. 8 shows a focus lens arrangement for three different image planesin an example embodiment consistent with the instant disclosure;

FIGS. 9A-C show a constant beam waist at the back aperture of a GRINendoscope, consistent with the present disclosure;

FIG. 10A shows beam scanning, consistent with an embodiment of theinstant disclosure, at a location slightly above the back aperture ofthe GRIN endoscope relay;

FIG. 10B shows that the central “chief rays” of the same embodiment ofFIG. 10A are parallel to the axis of the endoscope;

FIG. 11 shows only the chief rays of the same scan angles in FIG. 10;

FIG. 12 shows an example machined handheld device and integratedendoscope, in accordance with the instant disclosure;

FIGS. 13A-B show example embodiments of a handheld device, andcomponents thereof, consistent with the instant disclosure;

FIG. 14 shows a light path through an example embodiment of a handhelddevice, and the light paths relation to a photodiode for light powersensing;

FIGS. 15A-B show example embodiments of an integrated endoscope inaccordance with the instant disclosure;

FIG. 16 shows a light path through a needle package and a GRIN-basedendoscope in an example embodiment;

FIG. 17A shows an example imaging arrangement of the integratedendoscope relative to a set of muscle sarcomeres;

FIG. 17B shows a side view of the example imaging arrangement of FIG.17A;

FIG. 18 shows a constructed needle, in accordance with the instantdisclosure, having a pocket channel for GRIN optics;

FIG. 19 shows a tri-facet geometry of an example embodiment of a needle;

FIG. 20 shows suction line and GRIN optics placement in a needle in anexample embodiment of the instant disclosure;

FIG. 21 shows suction inlet geometry of an example embodiment of theintegrated endoscope;

FIG. 22 shows a signal wire connected to a needle arrangement of anintegrated endoscope in accordance with an example embodiment of theinstant disclosure;

FIG. 23A shows an example probe clamping mechanism for securing anintegrated endoscope to a handheld device based on the instantdisclosure;

FIG. 23B shows a perspective view of FIG. 23A;

FIG. 24 shows an integrated endoscope alignment based on the instantdisclosure;

FIGS. 25A-D show an example procedure, in accordance with the instantdisclosure, for clamping an integrated endoscope to a handheld device;

FIG. 26 shows a spring loaded trigger injector, in an exampleembodiment, that is used to deliver the integrated endoscope to a samplebeneath the skin;

FIG. 27A-B show an example embodiment of a dual movement lockingmechanism that prevents an integrated endoscope form being released froman injector;

FIGS. 28A-C show an example procedure for attaching a handheld deviceand integrated endoscope, consistent with the instant disclosure; to ahuman subject; and

FIG. 29 shows an example muscle image achieved through use of a handhelddevice and integrated endoscope, consistent with the instant disclosure,of a tibialis anterior muscle of a human subject.

While the invention is amenable to various modifications and alternativeforms, specifics thereof have been shown by way of example in thedrawings and will be described in detail. It should be understood,however, that the intention is not to limit the invention to theparticular embodiments described. On the contrary, the intention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention.

DETAILED DESCRIPTION

The present invention is believed to be useful for a variety ofdifferent thick-tissue evaluation applications, and the invention hasbeen found to be particularly suited for use in minimally-invasivemedical applications, arrangements and methods which are benefited fromhigh-resolution details of the thick tissue under examination. Asdiscussed in the background above, these biomedical approaches include,but are also not necessarily limited to, in vivo examinations. Variousaspects of the invention may be appreciated through a discussion ofexamples using this context.

In connection with various aspects of the present invention, it has beendiscovered that motion artifacts can be avoided by increasing the linescan rate as a function of the sarcomere dynamics and physiologicalmotion, and the ability to collect the responsive signals that arepredominantly present due to properties intrinsic to the structure. Asthese signals are typically weak and extremely difficult to detect, evenby the most sophisticated of microendoscopes, controlling the line scanrate can be important for high resolution images of biological structurein and around sarcomere. Consistent with an important aspect of variousembodiments of the present invention, unexpected high resolution imagesof this sarcomere-related structure are obtained by setting the scanrate to substantially minimize motion artifacts while permitting for thecollection of these weak intrinsic-based signals.

In a more specific embodiment, images of sarcomeres and sub-cellularstructures are obtained by using the optical probe to send light pulsestoward structure in the biological thick tissue at a sufficiently fastline-resolution rate to mitigate motion artifacts due to sarcomeredynamics and physiological motion. This rate being sufficiently fast tocause (in response to the light pulses) signals to be generated from andacross a sufficient portion of the structure to span a sarcomere length,and to collect selected aspects of the generated signals that arepredominantly present due to properties intrinsic to the structure.

Consistent with one example embodiment of the present invention, anapparatus permits for very-high resolution characterization ofcellular-level structure in thick tissue in a manner that is minimallyinvasive. The apparatus is capable of reverse direction imaging withoutstaining or otherwise introducing a foreign element used to generate orotherwise increase the sensed light. The apparatus has a probe thatincludes a light generator for generating light pulses that are directedtowards certain structures located within the thick tissue. The lightpulses interact with intrinsic characteristics of the tissue structuresto generate a signal. An emitted-light collector collects light (e.g.,excited light or light based on SHG) used to characterize aspects of thethick tissue. Reliance on intrinsic characteristics of the tissuestructures is particularly useful for applications in which theintroduction of foreign substances to the thick tissue is undesirable,such as in human imaging.

Consistent with another embodiment of the present invention, the lightgenerator and sensor are used in vivo using a probe, such as a needle orsimilar injection device, to minimize invasiveness while collectingsufficient cellular-level information for detailed visualizations. Invivo applications can be subject to image artifacts resulting frommovement of the target animal. The light is directed from the apertureof the probe to the targeted thick tissue. The probe includes a lightcollector with a light-directing lens arrangement designed to provide aprobe diameter that is sufficiently-small (in a specific exampleembodiment, about 1 mm to about 0.35 mm) to permit for needle-likeinsertion of the probe into the targeted thick tissue. For non-SHGapplications, the probe has an objective with a numerical aperture (NA)and other attributes adequate for collecting intrinsic-based signals.

In connection with the present invention, certain embodiments use aprobe to capture relatively low-intensity, intrinsically-based signalsfor a bright image with fine details of tissue structure, such assarcomeres, while being sufficiently-narrow to be implemented inneedle-like dimensions for microendoscopic applications performed invivo.

According to experimental example embodiments, the above-describedscopes have been implemented using three example sizes of gradientrefractive index (GRIN) lenses: 1000, 500 and 350 microns O.D. To fitinside a needle for endoscopic delivery, the followingcommercially-available needles can be used:

endo needle needle ID needle OD 1000 16-18 Ga 1070-1270 micron 1270-1650 microns  500 21-23 Ga 510-570 micron 640-820 microns 350 24-25Ga 370-410 micron 510-570 micronsSuch needles are available, for example, from Popper & Sons, New HydePark, N.Y.

In some applications, two-photon fluorescence microendoscope probes areimplemented with minimally invasive compound GRIN lenses with flexiblefiber-optic technology.

In another instance, the present invention is implemented in areverse-direction system using a light generator and sensor located inclose proximity. Proximity can be measured as either a spatial distanceor as an angle relative to the direction of the light generated by thelight generator. In one particular instance, the sensed signal is theresult of fluorescence generated from excitation of the cell structure(e.g., from the mitochondria). Because fluorescence is an isotropicphenomena, the light is equally dispersed. Accordingly, the angle of thesensor relative to the light pulses need not be a criticalconsideration.

In another instance, the sensed light is the result of the light pulsespassing through the cell structure and creating an SHG signal. The SHGsignal is dependent on the light pulses, and the direction of the lightpulses is relevant to the direction of the SHG signal. It has beendiscovered that an SHG signal can sometimes be classified into threecomponents including forward directed, backscattered and backwarddirected. A forward directed SHG signal includes the signal componentsthat continue in the direction of the light pulses. A backscattered SHGsignal includes the signal components resulting from scattering of aforward directed SHG signal such that the SHG signal travels towards thelight generator and sensor. A backward directed SHG signal includes thesignal components that are directed opposite the direction of theoriginating light pulses without scattering. Thus, the placement of thesensor affects the relative collection efficiencies of the SHG signalcomponents that are received. For instance, the placement of the sensorin the path of the backward directed SHG signal component can beparticularly useful in reverse-direction systems (e.g., by facilitatingthe sensing of both the backward directed SHG signal and thebackscattering SHG signal).

Turning now to the figures, FIG. 1A illustrates an endoscopic imagingsystem adapted to excite optical signals based on intrinsic thick tissuestructures and to collect intrinsic signals in response. The systemfacilitates high-resolution imaging of thick tissue in vivo.Femto-second laser pulses (e.g., 80-150 fs) are generated by a laser210. In a particular instance, the laser is a Ti-sapphire lasergenerating light with a wavelength around 700 nm to 1000 nm. Theparticular wavelength can be selected depending upon the application.Due to the frequency-doubling characteristic of intrinsic SHG signals,the frequency of the SHG signal is directly proportional to thefrequency of the excitation light (generated pulses). Thus, thegenerated pulses can be selected to minimize tissue absorption andscattering of the SHG signal for in vivo applications. Microendoscopicprobe 112 acts to both direct the generated pulses and collect theintrinsic signals. This means that for fluorescent and SHG signals, theimaging process relies primarily upon backward directed/scattered(low-energy) light.

For intrinsic fluorescent and SHG signals, the probe can be inserted inclose proximity to the targeted tissue or, as shown by muscle fiber 114,inserted into the targeted tissue. For isotropic light, the amount oflight collected by a given collector changes relative to the distancefrom the source. Moreover, absorption and scattering of light fromsurrounding tissue increases as the distance from the source increases(the scattering length is about five times shorter than the absorptionlength). For approaches directed to SHG signal generation, it has beendiscovered that the SHG signal collected by a probe aligned with theexcitation light generator (e.g., collecting backward SHG signals) andin close proximity to the thick tissue is surprisingly strong. This isparticularly useful for non-invasive and minimally-invasive in vivoimaging. The imaging time can also be increased to increase the totalamount of light received; however, increased imaging time can lead toincreased susceptibility to physiological motion resulting in unwantedmotion artifacts in the image.

Physiological motion, such as respiration and blood flow, arecompensated for using a number of techniques. Using one such technique,microendoscopic probe 112 is inserted into the thick tissue. The probe112 is small enough to allow physiological motion of the thick tissue tocause corresponding motion in the probe 112, while still capturing theimaging signals as discussed above. Thus, the effects of suchphysiological motion are mitigated by corresponding motion in the probe112. The amount of allowable physiological motion can be estimated fromthe desired image resolution. For example, subresolution physiologicalmotion would minimally affect subsequent image quality. The relevantamount of physiological motion is dependent upon the resolution of thedesired image. For instance, images having a resolution on the order ofa few micrometers are not substantially affected by physiological motionwhere the imaging is directed over a smaller span, e.g., much less thanabout 1-2 micrometers. Other factors that would affect the correlationbetween physiological motion and probe motion include the depth of thethick tissue, the length of the probe, and the stiffness of the opticalfibers and the physical properties of the thick tissue.

In one embodiment, microendoscopic probe 112 is connected to controlarrangement 102 using fiber cables and control lines for scanning,whereas in other embodiments physical separation is not provided (e.g.,by fiber cables). Control arrangement 102 includes various lightgeneration and detecting components controlled by a processor/computer106. In a particular instance, light generation block 210 (e.g., aTi:Sapphire laser) produces light pulses that are transmitted to probe112 using fiber optics, and photomultiplier tube (PMT) block 108receives light collected by probe 112 using fiber optics. The use of aflexible light-transport medium is useful because probe 112 can be movedindependently relative to the position of light generation block 210 andPMT block 108. Scanning mirrors 228 provide directional control over thelight pulses. Endoscope optics 230 directs both the transmitted lightpulses and the corresponding collected light.

As discussed above, to minimize the size of the probe, the probe shouldbe sufficiently small and the objective and related optical propertiesof the probe should be able to capture the intrinsic-based signals. In aspecific example, the probe is a gradient refractive index (GRIN)-lensmicroendoscopic probe used to provide a minimally-invasive mechanism forimaging such signals in thick tissue in vivo. In another more specificembodiment, rather than securing both the probe and the subject, theprobe can be allowed to move with the thick tissue. This freedom ofmovement is particularly useful for reducing motion artifacts due tophysiological motion. For many applications, it should be appreciatedthat the first priority is to be able to collect the intrinsic signals,and the second priority is to make the probe small enough withoutcompromising the first priority; however, there are applications, suchas where the target tissue is pre-exposed, that may be performed that donot necessarily have the small size requirement.

In one instance, the probe scans the thick tissue using a scanningdevice to direct light pulses toward the thick tissue. One such scanningdevice is a micro-electro-mechanical systems (MEMS) mirror. Scanningmirror control 104 provides signals to control the scanning device. Thesize of the scanning device is also a component of the overall size ofthe probe.

In one specific example application involving visualization of dynamicsarcomeres, a system as illustrated in connection with FIG. 1A is usedto obtain images based on line-scan speeds of about 2 kHz, with eachline being 256 pixels long and a dwell time of 2 μsec. During imaging,each line is approximately 0.512 msec. To prevent damage to the tissueby the laser, the power is limited to less than 50 mW incident at thesample. In other embodiments, the SHG signal is boosted by increasingpower, but too much power might damage the tissue. With a limit on thesignal being generated at the sample and using desired (or optimized)optics for practically collecting the largest fraction of that signal,the signal-to-noise ratio (SNR) becomes a limiting factor. In oneinstance, the line scan is 2 KHz while still maintaining a reasonableSNR. These parameters are adequate for providing images of skeletalsarcomeres. In order to image faster, the noise is decreased or thefraction of the SHG signal (that is collected) is increased. When usinga PMT, which has a relatively low noise, decreasing the noise further isnot practical. To increase the fraction of the signal being collected,the NA of the GRIN lenses can be increased, e.g., from an NA of 0.46 toan NA of about 0.7; such an increase increases the fraction of SHGcollected by a factor of about two. In another application, the dwelltime is reduced to about 1 μsec and the scan rate is increased to about4 kHz.

Other physiologic movements are caused by the heart beat and breathingwhich are about 1 Hz and 0.2 Hz respectively. By imaging at about 2 kHz,embodiments of the present invention allows for the collection of anentire image of 256 lines (0.13 sec) during a time when the animal isbetween heartbeats or breathing, thus practically eliminating motionartifacts. Assuming the same magnitude of signal is being generated fromthe skeletal muscle; applications of the present invention are usefulfor imaging a beating heart or organs in the thorax and abdomen.

FIG. 2 shows a flow diagram of a method for use with an endoscopicimaging system adapted to excite optical signals based on intrinsicthick tissue structures and to collect resulting intrinsic signals. Theflow diagram shows two main paths, one beginning with the Ti:Sapphierpulsed laser 210 and the other beginning with the stabilized subject 212(e.g., animal or human, typically in vivo). These paths show theoperation of the imaging probe and the preparation of the subject,respectively.

Referring to the path beginning with block 212, stabilizing the subjectto some degree can be important and useful for limiting movement thatwould interfere with medical oversight during the procedure as well aswith the production of high-resolution images. In accordance withsurgical procedures used as part of the present invention, such physicalrestraint optionally includes sedation (214) and/or conventionalphysical restraints (216) for limiting or controlling motion. At moredetailed levels of tissue characterization, physical restraints caninclude conventional restraints to physiological motion such as bylimiting blood flow (218-219) and respiration (220). In otherembodiments (alone or in combination with those discussed herein),respiration compensation can be accomplished by sedation, holding one'sbreath or through forced ventilation timed so that pauses in theventilation occur during the imaging process. Also consistent withvarious ones of the above embodiments, block 222 shows use of amicroendoscope that is sufficiently small so that when it is insertedinto the thick tissue, the microendoscope moves with the tissue therebymitigating the effect of the motion.

As will be discussed below, certain embodiments of the present inventionproduce high-resolution images of significant thick tissue structurewithout requiring significant compensation for such motion. Consistentwith the above-discussed aspects of the present invention and asdepicted by blocks 224 and 226 of FIG. 2, such motion artifacts can beavoided by increasing the line scan rate as a function of the sarcomeredynamics and physiological motion, and the ability to collect theresponsive signals that are predominantly present due to propertiesintrinsic to the structure. In this manner, sufficient intrinsic-basedsignals can be picked up by a commercial microendoscope for producinghigh-resolution images of structure in and around sarcomere, whilemitigating artifacts caused by the sarcomere and related physiologicalmotion.

In a more specific embodiment also according to the present invention,motion artifacts are mitigated as needed for the application at hand. Inthis manner, a computer-based digital imaging application usesconventional (e.g., standard-deviation) calibration techniques todiscern the quality of the process. Should the data processing indicatethat the sarcomere lengths cannot be discerned (i.e., insufficientresolution), the images are rejected as having unacceptable degrees ofmotion artifacts. The sarcomere length within an image is found by acomputer algorithm, e.g., Fourier transform, wavelet transform or fittedsine-wave, such that a confidence interval is also generated for themeasurements. In one application, an example threshold for an acceptablestandard-deviation is about 5% (confidence interval is compared to anarbitrary value; +/−5% is commonly used, to discriminate between imagesthat contain measurable sarcomere lengths from those that do not).Data-capture adjustments, whether automatically by the computer ormanually by the system user, are then made and/or further imagingefforts are repeated.

As examples of such detailed images obtained according to theseembodiments of the present invention, FIGS. 1B and 1C are respectivereproductions of images of sarcomere and sub-cellular structure. FIG. 1Bis an image taken from in vivo mouse lateral gastrocnemius using a 350μm endoscope. The scale bar indicates 25 μm. FIG. 1C is athree-dimensional reconstruction of lateral gastrocnemius muscle in aliving mouse. The model was created from a stack of 1 μm thick imagestaken with a 350 μm endoscope in a living mouse using SHG. The scale barindicates 10 μm.

With the microendoscope inserted in the subject 212, the laser 210initiates the methodology by sending pulses through the microendoscope230 while using a GRIN endoscope and excitation pre-chirping and opticalsignal processing as is conventional, as depicted at 215. Scanningmirrors 228 direct the pulses at the relevant tissue site, and dichroicmirrors are used to separate the excitation light from the emissionlight (block 231). The line-resolution rate is set sufficiently fastrelative to the motion artifacts expected due to factors such assarcomere movement and physiological motion. In a particular instance,the pulses are directed such that they capture the entire length of thesarcomere being imaged. Through the various techniques discussed herein,signals are generated in response to the pulsed light. These signals arecollected by the probe and used as part of the data for creating thedesired image. This process may be repeated as desired. For example, themultiple line scans may be used to generate larger imaging sections orto capture sequential images of the same sarcomere under differentconditions. In a particular instance, the effectiveness of a form oftherapy may be evaluated using images captured both before and aftertherapy is provided for the patient.

The path beginning with the pulsed laser 210 shows two approaches. Afirst approach (left path) involves fiber optics attached to amicroendoscopic probe and a second approach (right path) is implementedwithout fiber optics. The fiber approach involves a first step ofexcitation pre-chirping to compensate for transmission over opticalfiber (e.g., to reduce group-velocity-dispersion). The signal is thenconverted to the desired shape and path using conditioning optics androuted to polarization rotating optics. Upon polarization, the resultingfemtosecond laser pulses are passed through an optical excitation fiber232, such as a photonic crystal fiber. The second, non-fiber optics,approach operates much the same as the first approach without the needto compensate for transmitting the pulses through an optical fiber.

Once the optical pulses reach the microendoscopic probe, a scanningdevice (e.g., MEMS mirror) directs the pulses. A dichroic mirror allowslight of a certain wavelength to pass, while reflecting light ofanother. Thus, the dichroic mirror separates the laser pulses(excitation light) from the intrinsic signals (emission light). Thelaser pulses are directed through the GRIN microendoscope and to thethick tissue. The laser/excitation pulses striking the thick tissueresult in intrinsic signals (240A for intrinsic fluorescence (TPF) or240B for SHG). The GRIN microendoscope 230 collects the intrinsicsignals passing them to the dichroic mirror. The dichroic mirror routesthe collected signals towards emission filters 246. The collectedsignals pass through emission fiber 250 and routing optics 252 (or fiberoptics) to a photo-detector 254. The photo-detector 254 receives anddetects the collected signal. A processor 260 running customizablesoftware processes the information for producing the data 264 inresponse to the photo-detector and thereby permitting for structurevisualization. In one instance, the software compensates for motionartifacts and an image of the thick tissue is then generated forviewing.

In one particular embodiment, a microscope objective focuses ultrashortpulsed laser illumination onto the face of a gradient refractive index(GRIN) microendoscope. The microendoscope demagnifies and refocuses thelaser beam within the muscle and returns emitted light signals, whichreflect off a dichroic mirror before detection by a photomultiplier tube(PMT). A 350-μm-diameter GRIN microendoscope clad in stainless steel canbe used for minimally invasive imaging in the arm of a human subject.

For static imaging of individual sarcomeres, another embodiment providesimages of a single mouse muscle fiber in culture, acquired usingepi-detection of two-photon excited autofluorescence and band-passfiltered to highlight sarcomeres. As a variation, a band-pass filteredimage of the same fiber can be obtained using trans-detection ofsecond-harmonic generation (SHG). As an enhancement, overlaying theabove two image types reveals that autofluorescence signals, thought toarise from mitochondria located mainly at the Z-discs of sarcomeres,interdigitate with the SHG signal thought to arise in myosin tails.

Optical probe systems described herein can be implemented as amicroendoscope probing approach, according to the present invention, byusing very small lens systems having an acceptable objective lens andoverall diameters as described above. For instance, such microscopicendoscopes can be implemented using lens technology described in U.S.Pat. No. 5,161,063 and as described in other references including, butnot limited to, technology that is commercially-available from a varietyof manufacturers. One such manufacturer is Olympus (as cited in U.S.Pat. No. 5,161,063) which markets such scopes having diameters at about700 microns; other acceptable microscopic endoscopes can be similarlyconstructed using miniature-sized lens. For further informationregarding such systems, reference may be made to, “In Vivo Imaging ofMammalian Cochlear Blood Flow Using Fluorescence Microendoscope”,Otology and Neurotology, 27:144-152, 2006, “In Vivo Brain Imaging Usinga Portable 3.9 Gram Two-photon Fluorescence Microendoscope”, OpticsLetters, Vol. 30, No. 17, Sep. 1, 2005, and the following U.S. PatentPublications: No. 2004/0260148 entitled “Multi-photon endoscopic imagingsystem”; No. 2004/0143190 entitled “Mapping neural and muscularelectrical activity”; No. 2003/0118305 entitled “Grin fiber lenses”; No.2003/0117715 entitled “Graded-index lens microscopes”; No. 2003/0031410entitled “Multi-photon endoscopy”; No. 20020146202 entitled “GRIN fiberlenses”; and No. 2002/0141714 entitled “Grin-fiber lens based opticalendoscopes”.

In certain systems and applications of the present invention,embodiments described herein include optical fiber arrangements, and insome applications, a bundle of optical fibers. Various exampleembodiments are directed to the use of optical fibers such as thosedescribed in the following U.S. Patent Publications: No. 2005/0157981entitled “Miniaturized focusing optical head in particular forendoscope” (to Berier et al.), No. 2005/0207668 entitled “Method forprocessing an image acquired through a guide consisting of a pluralityof optical fibers” (to Perchant et al.), No. 2005/0242298 entitled“Method and equipment for fiber optic high-resolution, in particularconfocal, fluorescence imaging” (to Genet et al.) and No. 2003/0103262entitled “Multimodal miniature microscope” (to Richards-Kortum et al.);and as those described in the following U.S. Pat. No. 6,766,184(Utzinger et al.) entitled “Methods and apparatus for diagnosticmultispectral digital imaging,” U.S. Pat. No. 6,639,674 (Sokolov et al.)entitled “Methods and apparatus for polarized reflectance spectroscopy,”U.S. Pat. No. 6,571,118 (Utzinger et al.) entitled “Combinedfluorescence and reflectance spectroscopy,” and U.S. Pat. No. 5,929,985(Sandison et al.) entitled “Multispectral imaging probe”. Each of theseabove-cited documents is fully incorporated herein by reference.

Various embodiments of the present invention are specifically directedto measurement of sarcomere lengths in healthy subjects and inindividuals with neuromuscular diseases, allowing discovery of themechanisms leading to disabling muscle weakness. For example, in theclinic, the device is used as a diagnostic tool to determine the causeof weakness.

The capacity of muscles to generate forces is highly sensitive tosarcomere length. Muscles generate their maximum force at a sarcomerelength of approximately 3 μm, but generate almost no force at lengths of2 μm or 4 μm. In some instances, profound weakness in patients withneuromuscular diseases, such as cerebral palsy, may be caused by alteredsarcomere lengths. The ability to confirm this, in a variety of patientpopulations, enables important studies that examine the mechanisms ofmuscle weakness in persons with neuromuscular diseases.

In another example, implementations of the present invention are appliedduring surgery wherein this technology is used to set sarcomeres to theright length. The microendoscope is inserted into muscle in order tovisualize and measure the sarcomere lengths and the relatedmuscle-attachment points to provide maximum muscle strength followingmusculoskeletal surgeries. This approach is used to improve the outcomeof tendon lengthenings, tendon transfers, joint reconstructions andother musculoskeletal reconstructions.

Another application is directed to cardiac health. Cardiac health isdependent on contraction of cardiac muscle cells and imaging ofsarcomeres in a manner consistent with the above enables distinction ofhealthy and diseased or damaged cardiac tissue. The response to drugsmay increase or decrease contractility, and imaging sarcomere dynamics,as enabled here, allows these assessments in living subjects and invitro. In accordance with the present invention, the followingdiscussion is illustrative of cardiac uses and applications.

FIG. 3 depicts a heart 300 having a channel (or lead) 320 introduced tothe heart for the purpose of altering a condition of the heart,according to one embodiment of the invention. Lead 320 includeselectrodes 310-316 for delivering electrical stimulus or for sensingelectrical properties of the heart. Lead 320 may be introduced to theheart using a number of different techniques. In this instance, lead 320is shown as being introduced through the coronary artery. In oneinstance, a pacemaker device, located external to the heart, controlsthe electrical stimulus provided by electrodes 310-316. Images may betaken of myocardial sarcomeres using the various methods, systems anddevices described herein. These images may include myocardium sarcomereas accessed via the endocardium or epicardium. In these applications,the above-discussed microendoscopes can be used to obtain such imagesvia myocardium tissue accessible from areas outside the heart or, aswith lead 322 and optical probe 324, areas within the heart. In thelatter application, the same access port (e.g., the coronary artery) oranother access port may be used.

Using the above approach for cardiac stimulating/monitoring with relatedcardiac imaging, various specific applications are realized. In aparticular instance, images taken of the myocardial sarcomere withoutstimulus from the electrodes 310-316 are compared to images taken of themyocardial sarcomere with stimulus from electrodes 310-316. This may beparticularly useful in assessing the effectiveness of a particularcardiac treatment. In another instance, images of various cardiactreatments can be compared. For instance, the effects of dual (atrialand ventral) stimulus may be compared against ventral only stimulus. Inanother instance, the location, voltage and pulse duration of theelectrical stimulus may be varied to allow for a comparison of therespective myocardial sarcomere images. In other instances, damagedcardiac tissue can be imaged to ascertain the extent of the damage or toassess the effectiveness of a treatment of the damaged tissue.

In one embodiment, an input component is used to trigger the imagingtime. Such an input component may originate from a number of sources.For instance, QRS signals of the heart, such as those captured by anelectrocardiograph, may be used to trigger the imaging and/or as part ofthe system (e.g., using an EKG system concurrent with the imagingapproach illustrated in FIG. 1A). In another example, signalsoriginating from the pacemaker device can be used to trigger the imagingand/or capture the myocardium (via a pacing signal) while imaging thesarcomere and monitoring the effectiveness of the treatment. Suchimage-timing techniques can be useful for capturing images of myocardialsarcomere that correspond to natural heart function, captured heartcontractions (e.g., electrode induced), and the like.

In other embodiments, the heart may be altered using other techniquesand combinations of techniques. For instance, electrical stimulus neednot be administered using the electrode/lead configuration displayed inFIG. 3. Instead, any number of techniques may be employed. Other heartaltering therapies, such as drug induced alterations, may also benefitfrom the imaging of the myocardial sarcomere. For background discussion,reference can be made to any number of U.S. patents directed to cardiacmonitoring and cardiac therapy.

In another cardiac-imaging application, a specific embodiment of thepresent invention is directed to the system shown in FIG. 1A modified toinclude a lead within the microendoscope probe to provide myocardiumstimulus that is used concurrent with the above-described dynamicsarcomere imaging. For example, such microendoscope lead(s) can becombined with or within a single lead channel in which multiple signalsare transmitted via the same lead channel for stimulation and monitoringpurposes such as by modifying embodiments illustrated in U.S. Pat. No.6,208,886, entitled “Non-linear Optical Tomography of Turbid Media”(e.g., see FIG. 9 showing multiple (send/receive) fibers in samechannel).

Accordingly, this specific embodiment includes the system of FIG. 1Amodified such that a lead channel simultaneously delivers the electricalleads (commonly used for cardiac stimulus) along with multiple(send/receive) optical fibers. The microendoscope probes are then usedas described in connection with the above embodiments to providemyocardium stimulus and/or capture concurrently with the dynamicsarcomere imaging. By varying the timing, phases and power parameters ofthe myocardium stimulus, suspect (diseased) cardiac sarcomere can beviewed at detailed levels not previously recognized and therebypermitting patient-customized cardiac monitoring, therapy and/orpace-signal control for overall cardiac management.

FIGS. 4A-4D illustrate such example approaches in accordance with thepresent invention. In each instance, a lead channel simultaneouslydelivers the electrical leads (commonly used for cardiac stimulus) alongwith multiple (send/receive) optical fibers. As shown in FIG. 4A, thechannel 402 includes multiple nodes 404-408 at which electrodes 414(such as 312 of FIG. 3) and/or microendoscopic probes (such as 112 ofFIG. 1) access the myocardium. Where a node 404 includes both anelectrode 410 and a microendoscopic probe 412 (i.e., the end of probe112 of FIG. 1), the electrode can be implemented as a conductiveterminal at or immediately adjacent to the probe. By using multiple onesof such nodes, control circuitry (for the electrodes and/or the optics)can be selectively enabled so as to explore and access different areasof the myocardium tissue without necessarily repositioning the channel402. FIGS. 4B-4D illustrate various configurations for the channel 402and the corresponding location of the nodes 410-434, which may besuitable for different applications. For further discussion relating todifferent configurations of the channel, reference may be made to U.S.Pat. No. 5,181,511 entitled “Apparatus and Method for AntitachycardiaPacing Using a Virtual Electrode” (e.g., see FIGS. 5 and 6A-6F),which ishereby fully incorporated by reference.

Additional Experimental Efforts and Related Embodiments

Here, we report direct visualization of individual sarcomeres and theirdynamical length variations using minimally invasive opticalmicroendoscopy to observe second harmonic frequencies of light generatedin the muscle fibers of live mice and humans. We imaged individualsarcomeres in both passive and activated muscle. Our measurements permitin vivo characterization of sarcomere length changes that occur withalterations in body posture and visualization of local variations insarcomere length not apparent in aggregate length determinations.High-speed data acquisition enabled observation of sarcomere contractiledynamics with millisecond-scale resolution. These experiments evince invivo imaging to demonstrate how sarcomere performance varies withphysical conditioning and physiological state, as well as imagingdiagnostics revealing how neuromuscular diseases affect contractiledynamics. Further, with such in vivo measurements of individualsarcomeres, we learn precisely the normal operating range or variabilityof sarcomere length, how physiological regulation may adjust sarcomerelengths, and/or how sarcomere lengths are disrupted in disease.

In specific experimental embodiments, we use an optical microendoscopehaving gradient refractive index (GRIN) microlenses (350-1000 μmdiameter), to enter tissue in a minimally invasive manner and providemicron-scale imaging resolution. To facilitate studies in humans,certain embodiments avoid use of exogenous labels and rather explore thepotential for microendoscopy to detect two intrinsic optical signals.The first of these signals represents autofluorescence from nicotinamideadenine dinuclueotide (NADH) and flavoproteins, which are concentratedin mitochondria along sarcomere Z-discs. The other signal representssecond-harmonic generation (SHG), coherent frequency-doubling ofincident light, which occurs within myosin rod domains. Ourinstrumentation has used an upright laser-scanning microscope adapted topermit addition of a microendoscope 512 for deep tissue imaging (FIG.5). A microscope objective 516 coupled the beam from anultrashort-pulsed Ti:Sapphire laser into the microendoscope, to allowgeneration of two-photon excited autofluorescence and second-harmonicsignals. In both cases signal photons generated in thick tissue returnedback through the microendoscope 512 and were separated from theexcitation beam based on wavelength (FIG. 2).

We started investigations by imaging autofluorescence andsecond-harmonic signals simultaneously from cultured muscle cells. Thetwo signals were distinguishable by wavelength, the partial polarizationof SHG signals and their dependence on incident light polarization, andthe predominance of trans-(forward-propagating) over epi-detected(backward-propogating) SHG signals (Methods). With <30 mW of incidentlaser power, sarcomeres were readily apparent using either intrinsicsignal, especially after band-pass filtering the images to removespatial frequencies representing distance-scales outside the plausiblerange of sarcomere lengths (1-5 μm). Overlaid images of autofluorescenceand second-harmonic signals revealed that the two arise spatially out ofphase within sarcomeres, as expected if autofluorescence were to comemainly from Z-disc mitochondria and SHG from myosin rods.

For use in live subjects, we imaged based on epi-detected SHG andautofluorescence signals from the lateral gastrocnemius muscle ofanesthetized adult mice. Although SHG primarily arises in theforward-propagating direction, we hypothesized that in thick tissuethere would be sufficient backward-propagation to allow in vivomicroendoscopy, due to multiple scattering of photons that wereoriginally forward-propagating. We discovered that in vivo SHG imagingof sarcomeres was feasible by microendoscopy using illuminationwavelengths of ˜820-980 nm and generally led to better sarcomerevisibility than autofluorescence imaging (see Methods, supra). SHG is aneffective, endogenous contrast parameter that can be used to visualizesarcomeres in living subjects, and for subsequent imaging we used SHGand 920 nm illumination.

We further explored capabilities for imaging sarcomeres in anesthetizedmice. After inserting a microendoscope into the gastrocnemius, weregularly imaged large assemblies of individual muscle sarcomeres (n=23mice). Cardiac and respiratory movements often caused significant motionartifacts at image frame acquisition rates of <4 Hz, but at 4-15 Hzsarcomeres were readily identifiable within raw images. Insertion of themicroendoscope helped stabilize underlying tissue, reducing tissuemotion and enhancing image quality. To test the utility of our data, weperformed several illustrative analyses of muscle fiber structure inlive mammals.

First, we determined average sarcomere lengths and their variabilitywithin individual muscle fibers and between adjacent fibers.Uncertainties in measurements of average sarcomere length withinindividual fibers can be reduced to limits set by the inherentbiological variability, rather than by instrumentation, since thedistance spanned by a large, countable number of sarcomeres can bedetermined at a diffraction-limited resolution. Thus, with 20-50 nmsarcomeres often visible concurrently, our measurements of averagesarcomere length have ˜20-50 nm accuracy. In connection with theinvention, we discovered that individual sarcomere lengths can bevariable, with up to ˜20% variations within a ˜25-μm-diameter vicinity.The degree of local variability is likely influenced by passivemechanical inhomogeneities and could not be examined previously withouta technique such as ours for visualizing individual sarcomeres.

We created three-dimensional models of muscle fiber structure fromstacks of SHG images acquired at 0.5 μm depth increments within tissue.Construction of these models used the optical sectioning provided by SHGimaging which, like two-photon imaging, generates signals from aspatially restricted laser focal volume (see Campognola, P. J. et al.,“Three-dimensional High-resolution Second-harmonic Generation Imaging ofEndogenous Structural Proteins in Biological Tissues.” Biophy J 82,493-508 (2002)). We thereby verified that the muscle fibers we imagedwere almost exactly parallel to the face of the endoscope, thuspermitting us to make accurate sarcomere length determinations byimaging in the two lateral spatial dimensions (Methods).

We next measured sarcomere lengths at different body positions. In thegastrocnemius of anesthetized mice (n=7), sarcomere lengths depended onthe angle of the ankle, as shown in FIG. 6A, due to changes in totalmuscle length. Across mouse subjects, sarcomere lengths shortened from3.15±0.06 (s.e.m) μm to 2.55±0.14 μm during changes in ankle angle from70-170 degrees. This matches the operating range of 3.18-2.58 μm that weestimated based on a biomechanical analysis (Delp, S. L. et al., “AnInteractive Graphics-based Model of the Lower Extremity to StudyOrthopedic Surgical Procedures.” IEEE Trans Biomed Eng 37, 757-767(1990)) using measurements of muscle length, pennation angle, moment armlength, and an assumed optimal sarcomere length of 2.8 μm for a 120°ankle angle.

We further used such microendoscopy to capture the dynamics of sarcomerecontractions. Because these dynamics elapse over milliseconds, weperformed laser line-scans at 200-1000 Hz perpendicularly across rows ofsarcomeres undergoing changes in length. To induce muscle contraction inanesthetized mice, we electrically stimulated the gastrocnemius proximalto the site of microendoscopy (see Methods, supra). This triggered acontraction, which we visualized with ˜1-3 ms time resolution (FIG. 6B).Across multiple mice (n=5) in which the microendoscope was inserted asimilar distance from the ankle, mean sarcomere length was 3.05±0.02(s.e.m) μm prior to stimulation and 2.55±0.03 μm afterwards (FIG. 6C).Mean contraction speed peaked at 8.00±0.05 (s.e.m) μm s⁻¹ duringelectrical stimulation (FIG. 6D), which is within the range of maximumfibers responding in vitro to a chemical stimulus.

To demonstrate the applicability of microendoscopy to studies anddiagnostics in humans, we visualized individual sarcomeres within theextensor digitorum muscle of healthy human subjects (n=3). After placinga 20-gauge hypodermic tube into the extensor digitorum, we inserted a350-μm-diameter microendoscope through the tube and into the muscle. Thehypodermic was removed and the microendoscope held in place. Thesubject's arm was placed in a brace, immobilizing the forearm and wristbut leaving the fingers mobile. After commencing SHG imaging we are ableto visualize sarcomeres and their length fluctuations. Motion artifactswere often substantial but were reduced by bracing the limb. This tacticdoes not eliminate artifacts due to involuntary muscle twitching, whichcould only be overcome by increasing the laser-scanning speed to400-1000 Hz. Subjects were asked to move their fingers into fully flexedand extended positions. Systematic variations in sarcomere lengthbetween these two positions were evident from images across allsubjects, but each person exhibited slightly different ranges ofsarcomere operation. With fingers flexed, mean sarcomere lengths fromthree subjects were 3.15±0.03 μm, 3.30±0.01 μm and 3.25±0.05 μm (n=12,17 and 11 trials, respectively); with fingers extended these values were2.97±0.03 μm, 3.24±0.02 μm, 3.12±0.02 μm (n=10, 10, and 7 trials),illustrating our ability to determine how human sarcomere lengths dependon body posture. Subjects reported feeling only mild discomfort duringimaging sessions due to insertion of the microendoscope, indicating apotential suitability for eventual use during routine diagnostics ofhuman sarcomere function.

Growing evidence from tissue biopsies indicates sarcomere structure andlengths are altered in numerous neuromuscular disorders that result frommutations in sarcomeric proteins. Visualization of sarcomeres bymicroendoscopy can facilitate efforts to diagnose the severity of theseconditions, monitor progression, and assess potential treatments. Othersyndromes in which monitoring sarcomere lengths might inform treatmentchoices include geriatric muscle loss and contractures due to cerebralpalsy or stroke (see Plotnikov, S. V. et al., “Measurement of MuscleDisease by Quantitative Second-harmonic Generation Imaging,” Journal ofBiomedical Optics in press (2008), and Ponten, E., Gantelius, S. &Lieber, R. L., “Intraoperative Muscle Measurements Reveal a RelationshipBetween Contracture Formation and Muscle Remodeling,” Muscle Nerve 36,47-54 (2007). Combined SHG and two-photon microendoscopy of sarcomerelengths and fluorescent sensors or proteins in mouse models of diseasesis a scientific tool to aid in the understanding of muscle biology andpathophysiology. Intraoperative sarcomere imaging during orthopedicreconstructions or tendon transfer facilitates efforts by surgeons toidentify and set optimal sarcomere operating ranges. By reducingreliance on unproven assumptions, such as regarding the distribution ofsarcomere lengths, in vivo sarcomere measurements improve biomechanicalmodels that inform the understanding of human motor performance anddevelopment of rehabilitation technology, robotics, and prostheticdevices. For further discussion in this regard, reference may be made toLieber, R. L., Murray, W. M., Clark, D. L., Hentz, V. R. & Friden, J.,“Biomechanical Properties of the Brachioradialis Muscle: Implicationsfor Surgical Tendon Transfer.” J Hand Surg [Am] 30, 273-282 (2005),Delp, S. L. et al.,“OpenSim: Open-source Software to Create and AnalyzeDynamic Simulations of Movement,” IEEE Trans Biomed Eng 54, 1940-1950(2007), and Manal, K., Gonzalez, R. V., Lloyd, D. G. & Buchanan, T. S.,“A Real-time EMG-driven Virtual Arm,” Comput Biol Med 32, 25-36 (2002).

Optical degradation can occur when changing focal planes using a GRINlens. In an example embodiment, a telecentricaly oriented GRIN endoscopeis designed in a manner that allows a user of the endoscope to changethe focal plane while maintaining constant power, maintaining andmaximizing the resolution, and maintaining a constant magnification. TheGRIN endoscope, which is a doublet consisting of a relay and objectivelens, operates at infinite conjugates. In other words, collimated light,light whose rays are in parallel, in the endoscope is pivoted at theback aperture rather than focusing a point at the back of the GRIN lens.The focal plane of the GRIN endoscope of the instant embodiment can bechanged by the user via an afocal focusing mechanism. The afocalfocusing mechanism adds convergence or divergence to the normallycollimated light and shifts the focal plane at the sample. This isopposed to translating a focusing lens to change the image planes.Further, in another example embodiment, the afocal mechanism isconfigured and arranged to keep the beam waist constant at the backaperture of the GRIN endoscope. Therefore, the power of the lightdelivered to the sample does not fluctuate during focusing. Further, thecollimated light beam supplied to the GRIN endoscope can be providedsuch that the beam overfills the back aperture of the GRIN endoscope. Inthis manner, the endoscope delivers the maximum available resolution,irrespective of the instant focal plane of the endoscope.

In the embodiments described herein, the focal plane of a GRIN endoscopeaccepting collimated light, and that delivers a focused spot at thesample's plane, can be altered by changing the shape of the incominglight beam. Turning now to FIG. 7, which shows a GRIN endoscope inaccordance with an example embodiment of the instant disclosure. In anexample embodiment, a pair of lenses 700/710 is arranged in an afocalarrangement. The pair of lenses 700/710 is used to change the shape oflight beam provided to the GRIN endoscope 760. The example embodiment ofthe GRIN endoscope shown in FIG. 7 also includes a scan mirror 720, ascan lens 730, a tube lens 740, and a dichroic mirror 750. The lensclosest to the scanning mirror 720 is fixed, the fixed focus lens 710,while the lens nearest the light source, the mobile focus lens 700,moves on an actuated stage. When the mobile focus lens 700 translates,the light beam diverges or converges depending on the direction the lensmoved. The divergence and convergence of the light beam is shown in FIG.8. FIG. 8 displays the focus lens arrangement for three differentdesired image planes. The left lens of the pair is the mobile focus lens800, whereas the right lens is a fixed lens 810. The position of thelens shown at the top of FIG. 8, also labeled as position 1, is designedfor shallow imaging (less than 100 μm). The position of the lens shownin the middle of FIG. 8, position 2, shows the neutral location wherethe outgoing beam is unaltered and yields a focal depth of 100 μm. Theposition shown in the bottom of FIG. 8, position 3, shows the lensarrangement designed for deeper imaging (greater than 100 μm). Themobile lens 800 can be placed at any location within its range, allowingthe user to change the focal depth at the sample continuously between 0and 150 μm. In each of the lens positions shown in FIG. 8, the exitingbeam waist is constant within the focal plane of the fixed lens 810,regardless of the position of the mobile lens.

Turning again to FIG. 7 and also to FIG. 8, placing the scan mirror 720in the focal plane of the fixed lens 710/810 provides a constant beamwaist at the back aperture of the GRIN endoscope 760 because the scanlens 730 and tube lens 740 image the mirror to the same plane.Therefore, regardless of the position of the mobile lens 700/800, theintensity profile at the back aperture of the GRIN remains the same, andtherefore power delivered to the sample remains constant. Further, incertain embodiments, the beam waist is sized so that it is slightlylarger than the back aperture of the GRIN endoscope. As a result, thebeam diameter stays fixed, and light overfills the GRIN endoscope, whichdelivers a maximum available resolution for all focal planes. Moreover,in certain embodiments, when the focal lengths of the two focusinglenses, the mobile focusing lens 700/800 and the fixed focusing lens710/810, are different, this mechanism doubles as a beamexpander/de-expander.

In FIGS. 9A-C, the resulting beam waist at the back aperture of the GRINendoscope is shown for the same lens positioning in FIG. 8. FIG. 9Ashows the beam waist of position 1 shown in FIG. 8. FIG. 9B shows thebeam waist of position 2 shown in FIG. 8. FIG. 9C shows the beam waistof position 3 shown in FIG. 8. Also shown in FIGS. 9A-C is the tube lens910 and the dichroic mirror 900, shown in the example embodiment of theGRIN arrangement in FIG. 7. In each of the arrangements, the diameter ofthe beam waist at the back of the GRIN endoscope 920 is constant. Thiskeeps the power at the sample constant and the resolution at an optimallevel for all focusing positions. Although the above embodiment isdetailed with reference to a GRIN lens arrangement, the afocal focusingmethod described could be employed in any scanning microscope (e.g.,laser scanning microscope) to change focus of the microscope withoutphysically translating the objective.

The magnification of the GRIN endoscope can also change when shiftingthe focus to different planes. In certain instances, precisemeasurements require the user of an endoscope to record and track theinstantaneous magnification, and correct for that factor in themeasurement. This type of repeated scale corrections is an unnecessaryhindrance, which can be eliminated using the telecentric lensarrangement describe in the instant embodiment.

“Telecentric” describes a lens arrangement whose chief rays exiting theGRIN endoscope objective are parallel to the optical axis. The parallelnature of the chief rays is independent of focus position, therefore,the size of the image plane remains constant apart from the depth thatthe probe is imaging.

The optical path through the GRIN endoscope of the instant embodiment isshown in FIG. 10A-B. A cutaway view of the back end of the GRINendoscope is shown in FIG. 10A, where collimated light 1040 pivots nearthe back aperture of the relay lens 1000. A cutaway view of the frontend of the GRIN endoscope is shown in FIG. 10B, highlighting theobjective lens 1010, a 90° reflecting prism 1020 (modeled as a solidsquare of glass for simplicity), and a 100 μm thick layer of tissue1030. In FIG. 10A-B the double dashed lines indicate the location of thecutaway. The relay lens 1000 consists of a half pitch multiple andtherefore acts as a one-to-one imaging system, transmitting the pivotedcollimated rays from its back aperture to its front aperture. The angleof the pivoted rays exiting the relay is identical to the incoming anglefor even multiples of the half pitch length, and is reversed for oddmultiples. The objective takes this light and focuses it to a point inthe imaging plane. When the light entering the endoscope is pivoted, thefocal point generated by the objective lens translates within thesample, making it possible to generate a scanned image. When the scannedlight pivots at the front focal plane of the objective, the chief raysexit parallel to the optical axis, and there is no magnification effectthat results from changing the focus position. In order to achieve thelack of magnification effect (interference), rather than pivot the lightdirectly at the back aperture of the relay, the scan plane is shifted bya small amount (approximately 400 μm) relative to the back aperture ofthe relay lens. This shift makes the apparent scan plane reside at thefront focal plane of the objective, and eliminates the magnificationeffect. Alternatively, the length of the relay lens itself can bealtered to eliminate the magnification effect.

Also shown in FIG. 10 is a close up of the back of a GRIN endoscope inaccordance with an example embodiment of the instant disclosure.Further, FIG. 10 shows where the pivoting of the beam takes place(approximately 400 μm above the surface of the relay lens 1000). Thisshifts the plane of scanning downstream so that the beam pivots at thefront focal plane of the GRIN objective, shown in detail in FIG. 10B.The central “chief rays” of the different scanned angles are parallel tothe axis of the endoscope, and therefore no magnification changes willresult when shifting the focal plane.

The telecentric effect is further demonstrated in FIG. 11, which showsonly the chief rays of the same scan angles from FIG. 10 to highlightthe fact that they are parallel 1100 to the optical axis. Shown in FIG.11 is the front focal plane 1110 of the objective 1150. FIG. 11 alsoshows the ray trace of the chief rays scanning at 0 degrees (1140), 1.5degrees (1130) and 3 degrees (1120) at the scan mirror. All three areparallel to the center axis of the endoscope. Further, the pivot pointof the chief rays occurs at the front focal plane 1110 of the objective1150 which lies within the relay lens 1160.

The GRIN optics with the telecentric arrangement, the afocal focusingmechanism, and the scan mirror and accompanying optics, can beincorporated into a single imaging system. An imaging system, consistentwith the instant disclosure, is further described below. The descriptionof the system is separated into two parts: description of a handhelddevice (HD), and description of an integrated endoscope (IE). When an HDis combined with an IE, scanning and signal collection necessary togenerate images of deep tissue can be performed. An example embodimentof an IE connected with the HD is shown in FIG. 12.

An example embodiment of a handheld device (HD) can be seen in FIG. 13A.Description of the HD will often include reference to an IE, which willbe discussed subsequently in further detail. In the example embodimentshown in FIG. 13A, an HD 1300 is shown connected to an attachedintegrated endoscope (IE) 1310 via a precision clamping mechanism (PCU)1305. Also included in this example HD 1300 is a collimating unit 1345,for collimating light 1330 provided to the HD. The light 1330 isdelivered by a flexible, low dispersion fiber optic, most likely anair-core photonic chrystal fiber. Further, a focus stage 1325 isincluded to change the focal plane at the sample. The collimated andfocused light then is provided to a MEMS scanning mirror 1320, asdetailed above, which reflects the light towards a dichroic mirror 1315.The light is reflected by the dichroic mirror 1315 though an integratedendoscope (IE) 1310. The precision clamping mechanism (PCU) 1305, withtranslational adjustment, aligns the IE 1310 to the optical axis withinthe HD 1300 and to the scanning mirror plane.

Also included in the arrangement shown in FIG. 13A is an integratedpower sensor 1350, which is aligned with the optical axis within the HD1300. The integrated power sensor 1350 provides information regardingthe alignment and functional status of the proceeding optical elements.The integrated power sensor 1350 can be calibrated to indicate the powerdelivered to the sample while imaging.

These components can also be seen in FIG. 13B, which shows the HD priorto integration of the components. The collimating unit 1345 of the HD isshown at the top of FIG. 13B. The collimating unit 1345 collimates lightfrom a light source connected to the exterior of the unit. Thecollimated light would then pass through, assuming all the components ofthe HD are connected, the focusing unit 1325. A scanning unit 1355provides a base for the MEMS mirror that reflects the collimated lighttowards the dichroic mirror, through the scan and tube lens (asdescribed above with reference to telecentric focusing) and then towardsthe sample (as described above in FIG. 13A and further with reference toFIG. 14 below). Also shown in FIG. 13B are example embodiments of anintegrated endoscope 1310, and a probe-clamping mechanism 1305, bothdiscussed in further detail below. The collection unit 1360 is thecomponent of the HD that both houses the dichroic mirror, directinglight towards the sample, and additionally collects the light from thesample, and directs that light towards a signal fiber alignment unit1350.

Turning now to FIG. 14, which shows a light path through the HD, anintegrated power sensor 1480 utilized in an HD 1410 can constantlymeasure the amount of optical power transmitted through the optical path1400 of the HD 1410, as reflected off a MEMS mirror 1490 and provided toa dichroic mirror 1450, prior to entering the optics of the IE 1420. Theintegrated power sensor 1480 can be situated behind a dichroic mirror1450 in and HD arrangement 1410. The integrated power sensor 1480 isconfigured to measure the approximately 1% of the excitation light,termed leakage light 1440, that leaks through the dichroic mirror 1450,and the remaining 99% of the light 1430 reaches the sample. The leakagelight 1440 is directly proportional to the amount of light that isincident on the IE optics.

The integrated power sensor 1480 can consist of a photodiode 1470 inseries with a fixed value resistor. When light falls on the sensor, thephotons are converted by the photodiode 1470 into electric current, andby passing through the resistor, a voltage is generated that isproportional to the power of the incident light. The voltage measurementis provided via electrical leads 1460, and is monitored by, a highlysensitive multi-meter.

The integrated power sensor, as consistent with the instant disclosure,can provide valuable information during sample imaging. For example, thepower sensor can indicate the functional status of all the proceedingcomponents. The sensor reading is recorded at a benchmark power levelafter achieving optical alignment. When the reading at that same powerlevel decreases either gradually or rapidly, the power change indicatesthat an optical element has been damaged, or has fallen out ofalignment. Further, this indicator can be used to optimize the lasercoupling into the delivery fiber because the indicator gives a directreading of how much light is getting from the laser, through the fiber,and through the optics of the device. Moreover, the integrated powersample can be used as a real time indicator of the power delivered tothe sample while imaging. After an IE is aligned to the HD, laser poweris varied, and the power exiting the HD is correlated to the reading onthe power sensor. Using these readings, the operator will have knowledgeof how much power is present at the tip of the endoscope, and thereforeprevent tissue damage from excessive laser power.

An integrated endoscope (IE) has been developed that allows forpuncturing of thick tissue (e.g., muscle), and delivery of imagingoptics in the same step. The IE has additional features that make itpossible to collect both high quality static and dynamic images. Thebasic construction of the IE is seen in FIGS. 15A and 15B. The IE iscomposed of a GRIN based microendoscope and a rigid needle with acutting point 1510; an outer insulating jacket, and a solid base thatconnects to a handheld device (as described in detail above). The IEalso contains additional adjustment points to fine tune the alignmentwith the optical pathway (shown in further detail in FIGS. 23A-B andFIG. 24) within the HD. FIG. 15A also shows a wire for stimulation orsensing 1500, a part of the IE, that is connected to the needle withcutting tip and internal GRIN endoscope 1510. Further, the IE includes asuction port connection 1520 for blood removal. FIG. 15B shows anembodiment of the IE containing two ports: a blood removal suction port1520, and a second port 1540 for providing a fluid to the sample. FIG.15B additionally shows the separate elements of the needle 1510, whichis described in further detail with reference to FIG. 16 and FIGS.18-21.

The microendoscope, described in detail above and shown again in FIG.16, consists of a relay lens 1640, a more powerful objective lens 1630,and a 45-45-90 prism 1620. The prism allows for “side viewing” with theendoscope. The “side viewing” orientation provides multiple benefits.The tissue can be punctured directly with the IE itself Additionally,there is no need for a separate delivery cannula. Furthermore, bytranslating the IE axially, it is possible to image different fiberswithin the same injection.

The endoscope cannot directly image along its axis due to the IEincorporating a needle with a solid point 1650. The needle 1650 isdisplayed transparently in FIG. 16 to facilitate visualization of theinternal components and their spatial relationship to the needle. Theprism 1620 bends the focused light from the objective 1630 out to theside of the IE. A laser light 1610 is provided through the endoscope,and passes through the GRIN relay 1640, and the GRIN objective 1630,where it is reflected by the prism 1620 and creates a focused spot 1600outside of the IE. In this way, a sample positioned at the side of theIE can be visualized.

Turning now to FIGS. 17A and 17B, which show muscle fibers 1700positioned along the side of the IE. The muscle fibers make contact withthe outer surface of the IE 1720, putting them in direct contact withthe imaging optics. In FIG. 17A, the striped pattern represents thesarcomere pattern 1700. The focus 1730 exiting the prism, or excitationcone, is perpendicular to the axis of the muscle fibers 1700. From theside, the focus 1730 is in the fibers near the periphery. Translating(indicated by the arrows in FIG. 17B) the needle 1710 within the holeallows direct imaging of separate fibers without the need for multipleinjections.

Turning to FIG. 18, which shows an example needle 1810 that is used withthe IE. The needle shown in FIG. 18 is machined out of a straight wire.A channel 1800 is cut along the axis of the wire that allows the GRINoptics to fit inside with the prism at the end of the channel, and flushto the perimeter. The needle 1810 has a solid tip (shown in detail inFIG. 19) which can be machined into a tri-facet trocar geometry, whichis highly efficient at penetrating tissue. The three faces intersect atthree edges which act like blades to slice through tissue. Thisarrangement helps ensure that the needle punctures the fascia thatenvelops the muscle, and also minimizes patient discomfort since thereis less force needed to deliver the needle. The tri-facet trocargeometry can be replaced with an additional arrangement that minimizesdamage or distortion of the sample at the injection site. In anotherexample embodiment of the needle design, a hollow steel tube is used, asopposed to a manufactured wire, and a solid tip is attached. The solidtip can be manufactured into a cutting tip.

Turning now to FIG. 19, this figure shows a detailed arrangement of thethree faces of the needle 1900. The three faces of the needle 1900intersect in a symmetric 120 degree pattern. The facets are aligned suchthat the cutting edge formed by the bottom two facets lies within theplane 1920 that bisects the optics channel. This orientation minimizesthe likelihood that one of the cutting edges would slice the imagedmuscle fibers. Instead, the muscle fibers will run along the flat face,and reach the prism intact. Images taken of punctured muscle verify thatthe fibers at the imaging site are not damaged. Other tip geometriescould also be used for the solid tip. Also shown in FIG. 19 is thefocused spot 1910 relative to the three faces of the needle 1900.

FIG. 20 shows suction line and GRIN optics placement in a needle in anexample embodiment of the instant disclosure. The channel 2040 can bemachined with a standard endmill, which produces an extruded rectangularpocket. The channel 2040 is large enough for the prism to fit inside,which means that the round cross sections of the GRIN lenses 2010 do notfully fill it. This provides an area where suction lines 2000 can beplaced to remove blood. Additionally, the suction lines 2000 can be usedfor delivery of a liquid. For example, formulations can be injected tothe imaging site to either aid with data collection, or to produce someother observable effect. The formulations can be a drug to reduce pain,clotting, to produce sustained contractions, or to inhibit contractions.In an example embodiment, the suction lines 2000 are cast into placeusing a medical device grade epoxy and Teflon® coated wires. The coatedwires are placed in the gap between the GRIN endoscope 2010, and theside wall of the needle channel 2040. The coated wires run the length ofthe endoscope, and rest above the prism 2030 so that they exit theneedle channel near the end 2020. When the endoscope is secured inplace, epoxy fills the voids between the channel 2040 and the GRINlenses 2010, and encapsulates the coated wires. Epoxy does not stick tothe Teflon. Therefore, after the epoxy sets, the coated wires areremoved, leaving a clean tube that runs the length of the IE, thuscreating the suction lines 2000.

In certain embodiments, the suction lines are merged at a suctionconnector, therefore, both lines either provide suction or both deliverfluid. In other embodiments, the suction lines are independent of oneanother, therefore, one line could be used to inject saline, forexample, while the other provides suction to clean the image site.

In an example embodiment, wires that are 100 μm in diameter are used.The holes at the top end of the IE can be plugged later, and a connectoris secured to allow a small tube to connect to the IE, which draws bloodout.

Turning now to FIG. 21, a wire segment 2120 is attached at the end ofthe suction lines in order to avoid the suction lines coring out muscletissue when the IE is injected. The smooth suction inlets 2100 are shownrelative to the focused spot 2110. The smooth suction lines 2100 can beachieved, for example, by scraping by the epoxy until the wire 2120 isexposed, and polishing the arrangement clean. Tissue that contacts theinlets 2100 while the needle is being injected will run into the wire2120, and slide over without coring because the wire 2120 is round andsmooth. In another example embodiment, the wire segment is not used, andinstead a geometry of the suction site that prevents coring is utilized.

The needle-optics package is secured inside a polymer jacket thatelectrically insulates the endoscope. A wire is attached to the needleat the top of the IE that enables the user to monitor electric signalsat the image site, or to apply a voltage to the muscle and illicit alocal contraction. In FIG. 22, the outer jacket 2210 and the wire 2200are shown near the base of the IE. The wire attaches to the base of theneedle, so the tip of the IE is in electrical communication with thiswire. In another example embodiment, the needle can have multiple,isolated conducting pathways that extend to the tip. Therefore, theneedle can simultaneously excite contraction, and record electricalsignals, or a differential method of measuring signals can be utilized.

Incorporating a separate integrated endoscope (IE) into a small,all-in-one device, such as the handheld device (HD), is difficult due tothe need for precise alignment of the optics. The alignment procedure ofan IE and HD is described in detail with reference to the figures.Turning now to FIGS. 23A and 23B, the base of the IE is a large “button”2350 that has a trapezoidal cross section. The button 2350 can be madeout of brass or any acceptable metal substitute. The faces of the IEbutton 2350 are coupled to the clamping surfaces 2300 on the HD, andrigidly fix the IE to the Probe Clamp Unit body 2370. The“plane-line-point” clamping mechanism, described in further detailbelow, enables the IE to be taken on and off the HD, and return to thesame clamped position with very high precision allowing for sterilizingthe IE. The IE should first be aligned, and then autoclaved, as the IEshould be autoclaved between uses. The clamps 2300 on the HD can bemoved to provide coarse alignment of the IE to the optical path of theHD. In another example embodiment, the optics of the needle could bechanged to have a selection of different IEs with different opticalproperties. Therefore, the “plane-line-point” clamping mechanism wouldallow the user to swap the different IEs with different opticalproperties in and out at will without the need for individual alignment.

This clamping mechanism is referred to as the Probe Clamp Unit (PCU).Proper position of the IE with the HD is important for maintainingoptical alignment, which is directly related to the optical performanceof the HD. The clamping strategy aims to locate the IE relative to theHD with particularity. A body in space has six degrees of freedom, andtherefore can be fixed in a space by utilizing six points of constraint.A plane is defined by three points, a line by two, and a point by oneyielding six points in total. FIGS. 23A and 23B show the PCU 2370 and anIE that is clamped in place.

The bottom of the button 2350 rests against the base plane 2320 of thePCU 2370. The base of the PCU acts as a planar constraint since it ismachined flat. When clamped, the IE is constrained to translationswithin this plane. The line constraint exists in the form of a long pin2340 that contacts one of the angled surfaces 2330 of the IE base. Thelong pin 2340 is held in one of the clamp jaws 2300, and contacts the 45degree face 2330 on the button. This constrains the IE to translationsalong this line within the original plane. The flat side of the button2350 contacts a vertical pin 2310 which constrains the IE to a point onthe previously fixed line 2340, and therefore fully constrains the IE.

The function of the PCU is precision (not accuracy). In other words, thePCU mechanism positions an IE in a particular location, with a low levelof variation, but the location is not unique. The clamped location ofthe IE can be changed by translating either of the constraint clamps2300. Translating the constraint clamps alters the actual location ofthe clamped IE, but will not change the repeatability of the positioningin the new location. The translating constraint clamps can be utilizedto adjust the optical alignment between the HD and the IE prior toimaging. The location of the laser beam within the HD can certainlydeviate slightly from its ideal location. Therefore, when the laser beamreaches the IE, the optics within the IE may be slightly out ofalignment. The moveable clamps 2300 on the PCU 2370 allow the IE totranslate within the constraint plane, and maximize optical alignment.

To insure each individual probe is optimally aligned, the instantembodiment also allows for additional alignment freedom in the IE,because there is also variation within the IE itself. The base of the IE2350 allows additional alignment of the optics within the needle to theoptical path of the HD once the clamp positions are set. As shown inFIG. 24, the additional alignment can be accomplished by loosening andtightening the opposing pairs of set screws 2400/2410 to move theneedle-endoscope portion of the IE within the pocket of the IE base.Once optimal alignment is achieved, the needle-endoscope portion of theIE can be fixed rigidly to the base. Different IEs can be swapped in andout of the HD in any given order without the need for additionalalignment. This functionality allows for imaging multiple subjectsquickly.

In order to ensure accurate alignment of the IE and the HD, an exampleclamping procedure of the IE and the PCU is provided. Turning to FIGS.25A-D as a reference, a first clamping force is generated by the springloaded block 2500. When engaged, the springs within this mechanism pushan angled block 2505 against the IE base 2510, and generate a lateraland downward force that secures the IE relative to the HD.

The solid arrows of FIGS. 25A-D represent an applied forced inproportion to arrow size, and a dotted line indicates the translation ofthe components on which the dotted lines rest.

In order to secure the IE to the HD, turning to FIG. 25A, the springloaded block 2500 is retracted to make space for the IE base 2510, andposition the HD over the IE base 2510. As shown in FIG. 25B, gentlepressure should be applied on the spring loaded block 2500, which willsqueeze the IE base 2510 between the line constraint pin 2515, and theangled portion 2505 of the spring loaded block 2500.

While maintaining pressure on the spring loaded block 2500, slightpressure is applied to a push rod 2520 to translate the IE base alongthe pin line until it contacts the point constraint 2530 on the opposingclamp 2535, as shown in FIGS. 25B-C. Turning to FIG. 25D by way ofexample, while continuing to maintain pressure on both the spring loadedblock 2500 and the push rod 2520, the pressure on the spring loadedblock 2500 should be increased. The increased pressure applied to thespring loaded block 2500 will compress the springs until a latchengages, and fixes the spring loaded block 2500 in place. The springsprovide a consistent lateral clamping load that also pushes the IE base2510 down into the PCU base 2540 due to the angled surfaces 2545 of theIE base 2510.

In certain embodiments of the instant disclosure, a rapid injector isprovided to deliver the integrated endoscope (IE) to a sample (i.e.,muscle) of interest beneath the skin. The use of an injector yieldsrepeatable, clean, and less painful injections of the IE. As can be seenin FIG. 26, the injector consists of a spring 2620 loaded plunger thatconnects to the base of the IE prior to injection. A locking mechanism2630 prevents the IE from accidentally shooting out of the injector. Thelocking mechanism 2630 can be a dual movement locking mechanism.Further, the injector includes a threaded portion on the end of theplunger with a stroke limiting block 2600 which makes it possible toadjust the depth of needle injection. A trigger 2610 is provided torelease the spring 2620, and inject the needle.

FIG. 27 shows the dual locking mechanism in more detail. In the normalposition, as shown in FIG. 27A, the base of the knob 2700 prevents theangled clamp 2710 from pivoting so the IE 2720 cannot be removed. FIG.27B shows that the knob 2700 must be rotated clockwise 2730, and pusheddown 2740, to pivot the angled clamp 2710 and release the IE 2720.

An example procedure for imaging a human muscle with a handheld device(HD), including an integrated endoscope (IE), can be seen with referenceto FIG. 28A-C. An appendage of a human is first stabilized, and an areaof interest is identified, FIG. 28A. A needle is inserted into thesubject, FIG. 28B, and the handheld device, FIG. 28C, is attached andaligned (as described in detail above).

Clear muscle sarcomere images can be achieved through use of theattachment procedure. For example, a tibialis anterior muscle image isshown in FIG. 29.

In an example embodiment, consistent with the instance disclosure, amethod is described for imaging an aspect of biological tissue,including muscle, via a light-delivering optical probe (in thebiological tissue). The method is performed by using an objective,during focal plane changes, to focus the objective in the probe on thebiological tissue while lessening adverse image-quality degradation. Incertain specific embodiments, magnification effects are eliminatedduring focal plane changes. The method continues by using the opticalprobe to send light pulses toward structure in the biological thicktissue at a sufficiently fast line-resolution rate to mitigate motionartifacts due to physiological motion. In certain embodiments, the lightpulses overfill the optical probe to deliver maximum resolutionregardless of the focal plane changes, and in other embodiments, thelight pulses provided to the optical probe are collimated and pivoted ata back aperture of the optical probe. Using the optical probe, as partof the method described, causes, in response to the light pulses,signals to be generated from and across a sufficient portion of thestructure to span a sarcomere length. In using the probe select ones ofthe generated signals are collected, the signals are predominantlypresent due to properties intrinsic to the structure. As part of themethod, data is provided in response to the collected signals. The datais used for high-resolution imaging of said portion of the tissuestructure.

In certain specific embodiments of the method for imaging, the objectiveof the optical probe, used during focal plane changes, is characterizedas telecentric. Further, magnification of the optical probe remainsconstant during focal plane changes. Moreover, in other specificembodiments of the method of imaging, constant power and maximumresolution of the light pulses are maintained during the focal planechanges. Constant power is maintained by providing the light-pulses witha constant beam waist. Maximum resolution is maintained by providedlight-pulses to overfill a back aperture of the optical probe.

Another embodiment of the method uses an afocal lens arrangement to addconvergence or divergence to the light pulses thereby shifting the focalplane at the biological thick tissue. In those embodiments utilizing anafocal lens arrangement, the afocal lens arrangement includes a mobilelens and a fixed lens. In other embodiments of this method, the lightpulses provided to the optical probe are collimated and pivoted at aback aperture of the optical probe.

The instant disclosure is also directed towards an apparatus for imagingan aspect of biological tissue, which includes muscle. In certainembodiments, this apparatus can be used in a method of imaging an aspectof biological tissue (including muscle). The apparatus includes alight-delivering optical probe. The optical probe is configured andarranged with one end to be placed in the biological tissue. Thelight-delivering optical probe is further configured and arranged tosend light pulses toward structure in the biological thick tissue at asufficiently fast line-resolution rate to mitigate motion artifacts dueto physiological motion. Moreover, the light-delivering optical probe isdesigned to cause, in response to the light pulses, signals to begenerated from and across a sufficient portion of the structure to spana sarcomere length; and collect select ones of the generated signalsthat are predominantly present due to properties intrinsic to thestructure. The apparatus additionally includes a telecentric objectiveand a collector. The telecentric objective in the light-deliveringoptical probe is designed to lessen adverse image-quality degradationduring focal plane changes on the biological tissue by maintainingconstant magnification during the focal plane changes. The collector isconfigured and arranged to provide data in response to the collectedsignals for high-resolution imaging of said portion of the tissuestructure.

In certain specific embodiments of the apparatus, the optical probefurther includes an afocal lens arrangement that is designed to maintainmaximum resolution of the optical probe. In other specific embodiments,the optical probe can include an afocal lens arrangement configured andarranged to maintain constant power of the optical probe. Thetelecentric objective, in other embodiments of the apparatus for imagingan aspect of biological tissue, is designed to maintain constantmagnification of the optical probe. In other embodiments, the apparatusalso includes an afocal lens arrangement configured and arranged toprovide convergence or divergence to the light pulses.

The instant disclosure also details a method for imaging an aspect ofbiological tissue, including muscle, via a light-delivering opticalprobe in the biological tissue. The method of the instant exampleembodiment is utilized by providing the optical probe with a needle, theoptical probe and needle being integrated with an electro-mechanical endportion that is configured and arranged to puncture the tissue and whilein the tissue, electro-optically access the biological tissue. Incertain specific embodiments, the signals communicated between theelectro-mechanical end portion and the biological tissue are thoseoptically sensed for muscular contraction and/or for measuring theresulting sarcomere changes in response to electrically stimulating asanother inventive aspect. Moreover, in other embodiments, the signalscommunicated between the electro-mechanical end portion and thebiological tissue are to electrically stimulate. The method continues byusing the optical probe to send light pulses toward structure in thebiological thick tissue, and cause, in response to the light pulses,signals to be generated from and across a sufficient portion of thestructure in the biological thick tissue to span a sarcomere length.Further, in response to signals communicated between theelectro-mechanical end portion and the biological tissue, selected onesof the generated signals are collected, the generated signals arepredominantly present due to properties in the structure. The methodthen operates by providing data in response to the collected signals forhigh-resolution imaging of said portion of the tissue structure.

In certain specific embodiments, the light pulses are sent towardstructure in the biological thick tissue at a sufficiently fastline-resolution rate to mitigate motion artifacts due to physiologicalmotion. Another embodiment of the method of imaging an aspect ofbiological tissue is further characterized in that the signalscommunicated between the electro-mechanical end portion and thebiological tissue include signals which are electrically stimulatingsignals as well as the responsive optically sensible signals. Moreover,the responsive optically sensible signals are useful for detectingmuscular contraction and/or for measuring the resulting sarcomerechanges.

The needle of the optical probe used in the method can be, in certainembodiments, translated axially relative to tissue enabling multipleindependent measurements from a single injection. Further, in otherembodiments, the needle has differing optical properties for wide fieldor high resolution imaging.

The instant disclosure is also directed towards an optical imagingapparatus for imaging an aspect of biological tissue including musclevia an imaging-processing microscope and a light-delivering opticalprobe in the biological tissue. The optical imaging apparatus, in theexample embodiment now described, includes an optical probe with aneedle. The optical probe and needle are integrated with an end portionthat is designed to puncture the tissue, and while in the tissue,electro-optically access the biological tissue. The optical imagingapparatus further includes an engageable clamp-mechanism interface. Theengageable clamp-mechanism interface is configured and arranged toattach the optical probe with the imaging-processing microscope whilemaintaining optical alignment for imaging processing of the biologicaltissue.

Another embodiment of the instant disclosure is directed towards amethod for imaging an aspect of biological tissue (including muscle)using a light-delivering optical probe having an objective. The method,of an example embodiment consistent with the instant disclosure,operates by providing a portable device for processing optical-signaldata from the optical probe. Further, the method works by puncturing thetissue with the probe, and while in the tissue, collecting signals forhigh-resolution imaging of the tissue while lessening adverseimage-quality degradation by controlling and maintaining power level andlight-beam resolution for light pulsed through the light-deliveringoptical probe and objective. In certain specific embodiments, theportable device utilized in the method is hand-held.

The instant disclosure is also directed towards a method for imaging anaspect of biological tissue including muscle via a light-deliveringoptical probe having an objective. The method is characterized bypuncturing the tissue with the probe, and while in the tissue,collecting signals for high-resolution imaging of the tissue whilelessening adverse image-quality degradation by moving fluid near abiological-tissue image site for improving optical clarity via theobjective. In certain specific embodiments of this method, moving fluidinvolves removing blood from the biological-tissue imaging site, and inother embodiments, moving fluid involves providing saline to thebiological-tissue imaging site.

In certain specific embodiments, the method of imaging is furthercharacterized in that the optical probe is used to send light pulsestoward structure in the biological thick tissue at a sufficiently fastline-resolution rate to mitigate motion artifacts due to physiologicalmotion; cause, in response to the light pulses, signals to be generatedfrom and across a sufficient portion of the structure to span asarcomere length; and collect selected ones of the generated signalsthat are predominantly present due to properties intrinsic to thestructure. In this specific embodiment, the method provides data, inresponse to the collected signals, for high-resolution imaging of saidportion of the tissue structure.

Methods Summary

Instrumentation. In vivo imaging was performed on a laser-scanningmicroscope (Prairie) equipped with a wavelength-tunable Ti:Sapphirelaser (Mai Tai, Spectra-Physics) and adapted to accommodate amicroendoscope (see Jung, J. C. & Schnitzer, M. J., “MultiphotonEndoscopy,” Opt Lett 28, 902-904(2003), and Jung, J. C. Mehta, A. D.,Aksay, E., Stepnoski, R. & Schnitzer, M. J., “In Vivo Mammalian BrainImaging Using One- and Two-photon Fluorescence Microendoscopy,” JNeurophysiol 92, 3121-3133 (2004). In most SHG studies, we used920-nm-illumination. Epi-detected emission was band-pass filtered(ET460/50m, Chroma). A 10×0.25 NA objective (Olympus, PlanN) focusedillumination onto the microendoscope. Static images were acquired at512×512 pixels with 8 μs pixel dwell time. Line-scans were 256-512pixels long with 4 μs dwell time.

Animal Imaging. After anesthetizing adult C57bl/6 mice, we placed amicroendoscope inside or atop the muscle via a small skin incision. Weused 1-mm- and 350-μm-diameter doublet microendoscopes (Grintech),respectively exhibiting 0.48 and 0.4 NA and 250-μm- and 300-μm-diameterworking distances in water.

Human Imaging. Under sterile conditions, a stainless steel clad350-μm-diameter microendoscope was inserted into the proximal region ofextensor digitorum via a 20-gauge hypodermic. We used a 350-μm-diametermicroendoscope (Grintech) with a 1.75 pitch relay and a 0.15 pitchobjective of 0.40 NA and 300-μm-working distance. In one situation,line-scan images were acquired at 488 Hz in the exterior digitorummuscle of a human subject with digits of the hand flexed and/orextended.

Data Analysis. Mean sarcomere lengths in static and dynamic images werecomputed in Matlab (Mathworks) by calculating the autocorrelation acrossan image region that was one pixel wide and parallel to the musclefiber's long axis. An 11^(th)-order Butterworth band-pass filterselective for 1-5 μm periods was applied to the autocorrelation. Fittinga sine to the resultant yielded the dominant periodicity and meansarcomere length. Analysis of length variations relied on measurement ofindividual sarcomere lengths performed at each pixel by findingdistances between successive intensity peaks along a line parallel tothe fiber's long axis. Locations of these peaks were found by fitting aone-dimensional Gaussian to each high-intensity region. A 2×5 pixelmedian filter, with its long axis aligned to the muscle fiber, smoothedthe resultant image of sarcomere lengths.

Methods

In vitro imaging. Single muscle fibers were prepared by enzymaticdissociation of tibialis anterior from C57bl/6 mice using a methodmodified from Carroll et al. Tibialis anterior from a freshly sacrificedadult C57lb/6 mouse was incubated in 0.2% collagenase (Sigma, type IV)solution for 3-4 hours. After incubation, single fibers were obtained bytrituration with a wide-mouth pipette, transferred to 90% Ringer'ssolution (in mM, 2.7 KCl, 1.2 KH₂PO₄, 0.5 MgCl₂, 138 NaCl, 8.1 NaHPO₄,1.0 CaCl₂; pH 7.4) and 10% fetal bovine serum, and incubated for <1 day.The imaging system comprised a custom laser-scanning microscope equippedwith a wavelength-tunable, ultrashort-pulsed Ti:Sapphire laser(Spectra-Physics, Mai Tai) and a 40× water 0.80 NA objective (Olympus,LUMPLFL). 720-nm-illumination was used to generate autofluorescence thatwas collected in the epi-direction and filtered with BG40 colored glass(Schott). 920-nm-illumination was used to generate SHG that wascollected in the trans-direction by an identical 40× water microscopeobjective and filtered by an ET460/50m filter (Chroma). In someexperiments using SHG, the polarization of the laser light was variedwith a half-wave plate to verify polarization dependence or to optimizesignal intensity. Acquired images were four frame averages of 512×512pixels using an 8 μs pixel dwell time.

Animal imaging. All animal procedures were approved by the StanfordInstitutional Animal Care and Use Committee. Adult C57bl/6 mice wereanesthetized by injection of ketamine (0.13 mg/g) and xylazine (0.01mg/g i.p.). The hindlimb was shaved and fixed to a frame such that jointangles could be controlled. The imaging site was periodically irrigatedwith Ringer's solution. In experiments on sarcomere dynamics, westimulated the muscle supra-maximally using a muscle stimulator(Medtronic, model 3128) with tungsten wires surrounding the proximaltibial nerve, which innervates the lateral gastrocnemius. We generallyused either a 1-mm-diameter doublet microendoscope (Grintech, GmbH),composed of a 0.75 pitch Li-doped gradient refractive index (GRIN) relaylens of 0.2 NA coupled to a 0.22 pitch Ag-doped GRIN objective lens of0.48 NA and 250-μm-working distance in water, or a stainless steel clad350-μm-diameter doublet microendoscope (Grintech, GmbH), composed of a1.75 pitch Li-doped GRIN relay lens of 0.2 NA coupled to a 0.15 pitchAg-doped GRIN objective lens of 0.40 NA with a 300-μm-working distancein water. We performed laser line-scanning by first acquiring areference image in two spatial dimensions and then choosing a linearpath parallel to the long axis of the fiber for subsequentline-scanning.

Model of sarcomere length versus joint angle. The change inmuscle-tendon length (dl^(mt)) with change in ankle joint rotation angle(dθ) was determined using:

$\begin{matrix}{{\frac{d\; l^{mt}}{d\;\theta} = {ma}},} & \;\end{matrix}$where ma is the moment arm of the muscle. The moment arm and itsvariation with joint angle were determined by calculating the distanceto the joint's center of rotation along the direction normal to themuscle's line of action. We calculated the change in muscle fiber length(dl^(m)) with change in ankle angle during passive motion by assumingthat tendon stretch was negligible and thus:

${\frac{d\; l^{m}}{d\;\theta} = {{ma}\left( {\cos\;\alpha} \right)}},$where a is the pennation angle of the muscle fibers. Once the change inmuscle fiber length with ankle angle was computed, the change insarcomere length (dl^(s)) with joint angle (FIG. 3) was estimated using:

$\frac{d\; l^{s}}{d\;\theta} = {m\;{acos}\;{\alpha\left( {l_{o}^{s}/l_{o\;}^{m}} \right)}}$where the optimal muscle fiber length (l^(m) _(o)) was determined bymeasuring the fiber length at the resting joint angle. The sarcomerelength at the optimal fiber length (l^(s) _(o)) was assumed to be 2.8μm.

Human Imaging. All human imaging procedures were performed in accordancewith FDA guidelines for the protection of human subjects (21 CFR 50) andapproved by the Stanford Institutional Review Board. Subjects' forearmswere restrained in a brace and fixed to the microscope'svibration-isolation table. All optical components were identical tothose used during animal studies. However, all components, includingmicroendoscopes and mounting components, contacting or potentiallycontacting human subjects at the imaging site were sterilized byautoclaving. After insertion of the microendoscope, subjects were askedto flex and extend their fingers and changes in sarcomere length weremonitored. Duration of testing was <60 minutes in all cases.

Data Analysis. Band-pass filtered images of sarcomeres were computedfrom raw images by applying an 11^(th)-order Butterworth filter thatacts as a band-pass for spatial periods between 1-5 μm. All analyzedimages contained between 20 and 50 sarcomeres. For each muscle fiber,average sarcomere length was determined along each of a series ofparallel lines aligned with the axis of the fiber. We report the meanand s.e.m. of this collection of measurements. Determinations ofaccuracy in average sarcomere length measured along a single line usedthe 95% confidence interval generated by a nonlinear least-squares curvefitting algorithm (Trust-Region algorithm, nonlinear least-squaresmethod). All data analysis was done in Matlab (Mathworks).

Assessment of sarcomere visibility. The intensities of epi-detected SHGand autofluorescence signals are influenced by severalwavelength-dependent processes, including attenuation of illumination inthick tissue, generation of signal photons at the focal plane,scattering of signal photons, and attenuation of signal photons withinthe detection pathway. Both the spatial arrangement and the contrastratio between the maximum and minimum signal intensities observed withinindividual sarcomeres also influence sarcomere visibility. Afterexploring the illumination wavelength range of 720-980 nm using ourtunable Ti:Sapphire laser, we found that given this light source and thetransmittance characteristics of our microscope, SHG imaging withillumination of ˜920 nm was most effective at revealing sarcomeres invitro. We do not claim that 920 nm is the optimum excitation wavelengthfor imaging sarcomeres in thick muscle tissue, but rather that SHGimaging with 920-nm-illumination permits characterization of sarcomerelengths and dynamics in live subjects.

Potential measurement errors. To minimize chances of photo-damage duringimaging we maintained incident laser illumination below 30 mW, areported approximate threshold for tissue damage. We also monitored forany physical signs of damage in the tissue. If a component of a musclefiber or its lateral inter-fiber connections were substantially damaged,one might expect to see punctate, local differences in sarcomerestructure distinct from surrounding tissue. We did not observe sucheffects, but rather observed sarcomeres with relatively uniform andsmoothly varying lengths. We also performed control studies in which wetested quantitatively for any differences in sarcomere lengths betweenpaired measurements obtained just prior to and then immediately afterinsertion of the microendoscope into the muscle. Prior to insertion wemeasured sarcomere lengths in the unperturbed muscle using an airobjective (Olympus, 20×, 0.4 NA, LMPlanFL). We then inserted amicroendoscope into the same tissue site and measured sarcomere lengthsagain. Comparison of the paired data sets revealed that sarcomere lengthdeterminations were virtually identical under the two conditions,differing by only 3.8±2.4% (mean±s.d.; n=45 measurement sites) andthereby precluding any substantial errors due to microendoscopeinsertion.

Another potential source of measurement error is parallax due tomisalignment of the microendoscope's optical axis relative to the musclefibers' transverse planes. However, a measurement error of just 1% wouldrequire a misalignment of over 8 deg, which was not observed in ourthree-dimensional data sets acquired with the microendoscope placed atopthe muscle. In the mouse lateral gastrocnemius we found that musclefibers were nearly parallel to the face of the microendoscope. Fromthree-dimensional image sticks we measured an average misalignment of3.3±1.8 deg (mean±s.d.; n=37 measurements from 4 stacks acquired in 4mice). Such consistent mechanical alignment probably results in part dueto pressure from the microendoscope on the muscle fibers. We concludethat in the lateral gastrocnemius measurement errors due toorientational misalignment are usually negligible. Similarly,misalignment errors seem likely to be minor in muscles in which thefibers lie parallel to the surface of the muscle, but perhaps less so inmuscles in which the fibers vary significantly from this orientation.

For discussion relating to the above embodiments, reference may be madeto the appendix document (Appendix A) filed in the underlyingprovisional patent application and entitled, “Direct Observation OfMammalian Sarcomere Extension In Skeletal Muscle Using MinimallyInvasive Optical Microendoscopy.” This appendix document and all otherpatent and non-patent documents cited herein are incorporated byreference, each in its entirety.

The various embodiments described above are provided by way ofillustration only and should not be construed to limit the invention.Based on the above discussion and illustrations, those skilled in theart will readily recognize that various modifications and changes may bemade to the present invention without strictly following the exemplaryembodiments and applications illustrated and described herein. As anexample, technology other than GRIN-lens technology may be used inimplementing the microendoscopes discussed above. As another example,the above-described methods and arrangements for using lead channelshaving multiple optic probes are applicable to both skeletal sarcomereand cardiac sarcomere. Such modifications and changes do not depart fromthe true spirit and scope of the present invention, which is set forthin the following claims.

What is claimed is:
 1. An apparatus for imaging a biological tissue of asubject, the apparatus comprising: a light source that is configured toprovide light pulses; a scanning mirror in optical communication withthe light source; an afocal lens arrangement including a fixed lens,wherein the afocal lens arrangement is configured to shift a focal planeat the biological tissue and is disposed between the light source andthe scanning mirror, wherein the light source, scanning mirror andafocal lens arrangement are configured and arranged to direct the lightpulses toward the biological tissue at a sufficiently fastline-resolution rate to mitigate motion artifacts due to contractilemotion or physiological motion of the subject, which light pulses causesignals to be generated from and across at least a portion of thebiological tissue; an objective in optical communication with thebiological tissue and the light source, wherein the objective isconfigured and arranged to (i) focus the light pulses from the lightsource to the biological tissue, and (ii) collect the signals in amanner that lessens adverse image-quality degradation during changes ofthe focal plane at the biological tissue; a collection member, includingcircuitry, in optical communication with the biological tissue and theobjective, wherein the collection member is configured and arranged tocollect select ones of the generated signals that are present due toproperties intrinsic to the biological tissue; and a computer processorthat is coupled to the collection member, wherein the computer processoris configured to generate an artifact-mitigated image of the biologicaltissue by at least (i) directing the light source to provide the lightpulses, (ii) directing the scanning mirror to send the light pulsestoward the biological tissue at a line scan rate of at least 1 kHz, and(iii) using the select ones of the generated signals collected by thecollection member to generate the artifact-mitigated image of thebiological tissue.
 2. The apparatus of claim 1, wherein at least aportion of the generated signals are second harmonic generation (SHG)signals, and wherein the computer processor is configured to image thebiological tissue using the SHG signals.
 3. The apparatus of claim 1,further comprising a probe in optical communication with the lightsource, wherein the probe is flexible.
 4. The apparatus of claim 1,further comprising a probe in optical communication with the lightsource, wherein the probe is integrated with an electro-mechanical endportion that is configured and arranged to contact the biological tissueand, while contacting the biological tissue, electro-optically accessthe biological tissue.
 5. The apparatus of claim 4, wherein the probe isa blunt probe or a needle.
 6. The apparatus of claim 1, wherein theobjective is a telecentric lens arranged in a light path in which thelight pulses travel and is configured to maintain constant magnificationduring the changes of the focal plane.
 7. The apparatus of claim 1,wherein the afocal lens arrangement is configured to maintain constantpower of the light pulses generated from the light source.
 8. Theapparatus of claim 1, wherein the afocal lens arrangement is configuredto maintain maximum resolution of the light pulses.
 9. The apparatus ofclaim 1, wherein the afocal lens arrangement comprises a mobile lens,wherein the mobile lens is configured to shift the focal plane at thebiological tissue.
 10. The apparatus of claim 1, wherein the afocal lensarrangement is configured to provide convergence or divergence to thelight pulses generated from the light source.
 11. The apparatus of claim1, wherein the objective is a lens configured to maintain constantmagnification of the light pulses.
 12. The apparatus of claim 1, whereinat least a portion of the generated signals are autofluorescencesignals, and wherein the computer processor is configured to image thebiological tissue using the autofluorescence signals.
 13. The apparatusof claim 4, wherein the probe is configured to translate axiallyrelative to the biological tissue.
 14. The apparatus of claim 5, whereinthe probe is a needle that is configured to puncture the biologicaltissue.
 15. The apparatus of claim 5, herein the probe is a blunt probethat does not puncture the biological tissue.
 16. The apparatus of claim5, wherein the probe is a needle that is configured to translate axiallyrelative to the biological tissue and to permit multiple independentmeasurements from a single injection.
 17. The apparatus of claim 5,wherein the probe is a needle that has differing optical properties forwide field or high resolution imaging.
 18. The apparatus of claim 6,wherein the objective is a lens configured to maintain constantmagnification of the light pulses.
 19. The apparatus of claim 1, furthercomprising an engageable clamp-mechanism interface that attaches aremovable probe to the apparatus while maintaining optical alignment forimage processing of the biological tissue.
 20. The apparatus of claim 1,wherein the image of the biological tissue corresponds to at least 256pixels spanning immediately adjacent z-lines of noncontracted sarcomerein the subject.
 21. The apparatus of claim 1, further comprising anintegrated power sensor in optical communication with the light source,the scanning mirror, and the afocal lens arrangement.
 22. The apparatusof claim 21, wherein the integrated power sensor comprises a photodiodeand a resistor.
 23. The apparatus of claim 22, wherein the photodiode isconfigured and arranged to detect leakage light through a dichroicmirror.
 24. The apparatus of claim 1, wherein the computer processor isfurther configured and arranged to regulate a power level of the lightpulses at the biological tissue to prevent tissue damage.
 25. Theapparatus of claim 1, wherein the scanning mirror is disposed in a focalplane of the fixed lens.